Alleles on locus chromosome 4B from different parents confer tiller number and the yield-associated traits in wheat

Pleiotropy is frequently detected in agronomic traits of wheat (Triticum aestivum). A locus on chromosome 4B, QTn/Ptn/Sl/Sns/Al/Tgw/Gl/Gw.caas-4B, proved to show pleiotropic effects on tiller, spike, and grain traits using a recombinant inbred line (RIL) population of Qingxinmai × 041133. The allele from Qingxinmai increased tiller numbers, and the allele from line 041133 produced better performances of spike traits and grain traits. Another 52 QTL for the eight traits investigated were detected on 18 chromosomes, except for chromosomes 5D, 6D, and 7B. Several genes in the genomic interval of the locus on chromosome 4B were differentially expressed in crown and inflorescence samples between Qingxinmai and line 041133. The development of the KASP marker specific for the locus on chromosome 4B is useful for molecular marker-assisted selection in wheat breeding. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05079-4.


Introduction
Wheat (Triticum aestivum L.) is a long-historically grown crop in more than 40 countries.It feeds about 30% of the world's population.Despite China, as a leading wheat grower, producing approximately 17% of the global wheat, the demand for wheat is increasing due to the population growth and rapid urbanization [1][2][3].Increase in wheat yield per annum from the 1920s was estimated to be 1.29% and 1.50% for north and south winter wheat in China [4].Nevertheless, this yield increment does not meet the future demand for wheat [5].Further increase in grain yields is the highest priority in most wheat breeding programs throughout the country.
Wheat yield is determined by many agronomic traits, such as the number of spikes per unit area, grain numbers per spike (GNS), and thousand-grain weight (TGW) [6].Because direct selection of grain yield is difficult, the improvement of yield-associated traits is often conducted instead.In fact, GNS and TGW have been increased in wheat cultivars released in the past several decades in China [4].Many genetic loci governing grain weight were identified on different wheat chromosomes [7].Several studies reported quantitative trait loci (QTL) for grain weight on the short arm of chromosome 4B.QTKW.caas-4BS for grain weight was mapped in a 483 kb genomic interval in Doumai, which harbors three genes ZnF, EamA, and Rht-B1 [8].The functions of Rht-B1 (encoding gibberellin signaling repressor) and ZnF-B (encoding a RING-type E3 ligase) on grain yield were determined using natural deletion of a haploblock (∼ 500 kb) in wheat cultivar Heng597 [9].Grain weight is associated with grain size measured by grain length (GL) and grain width (GW) [10][11][12][13].Chen et al. (2020) [14] identified 30 stable QTL including QTgw.cau-7D and QGw.cau-7D for grain size on chromosome 7D.
A wheat spike is composed of spikelets borne on its rachis.Spike length (SL) is closely correlated with spikelet number (SN).Both traits are influenced by interaction between genetic and environmental factors.Li et al. [24] mapped two major QTL for SL, QSc/Sl.cib-5A and QSc/Sl.cib-6A, explaining 7.13-33.6% of the phenotypic variations.TaAPO-A1 confers total SN in European winter wheat cultivars [25].QSns.sau-2D was detected on the chromosome arm 2DS and can explain 10.16-45.68% of phenotypic variations [26].An important pleiotropic QTL, Q.SpnN/SpkLng/PH/SPP.3A, was identified on chromosome 3A.It was found to be associated with spikelet number per spike, spike length, plant height, and spikes per spike [27].
Some wheat cultivars have awns in glumes.Awn is an important organ for respiration and photosynthesis of spikes.It not only plays a role in protection and transmission of seeds, but also impacts on grain yield.Assimilates synthesized by wheat awns are transported to grains nearby [28].It is believed that B1, B2, and HD are the main genes inhibiting the awn development.Locus B1 (tipped 1) on chromosome 5AL regulates the phenotype of tip awns, and B2 (tipped 2) on chromosome 6BL shortens the awn length [29,30].Hd, a dominant allele of knotted 1 (kn-1) on the chromosome arm 4AS, regulates the hook-awn phenotype of wheat [31].
Wheat landrace Qingxinmai is featured by a large number of tillers.It can develop 30-40 spikes per plant.Breeding line 041133 develops large spikes and grains.Incorporation of favorable alleles of loci for tiller number, spike, and grain traits from Qingxinmai and line 041133 is an option to increase yield of wheat.Toward this end, dissection of QTL for the traits of interest is a prerequisite.A recombinant inbred line (RIL) population was developed from a cross between these genotypes.The purpose of this study was to unravel the genetic control of the agronomic traits with the aid of bulk segregant analysis-RNA-Seq (BSR-Seq), bulked segregant exome capture sequencing (BSE-Seq), and a wheat 16 K genotyping by target sequencing (GBTS) single nucleotide polymorphism (SNP) array.

Phenotypic performances
Qingxinmai and line 041133 differed significantly in tiller traits, total tiller number (TN) and productive tiller number (PTN), spike traits, spike length (SL), spikelet number per spike (SNS), and awn length (AL) (including top, central, and bottom positions on spikes), and grain traits, thousand-grain weight (TGW), grain length (GL), and grain width (GW) (P < 0.05).Qingxinmai had more tillers and longer awns, while line 041133 had longer spikes and larger kernels in different field trials (Fig. 1; Table 1).The broad-sense heritability (H 2 ) for these traits ranged from 0.40 to 0.99.
Variations were observed in traits of the Qingxinmai × 041133 RIL population investigated.The coefficients of variation (CV) of the tiller and spike traits (i.e., TN, PTN, SL, SNS, and AL) was greater than that of the grain traits (i.e., TGW, GL, and GW) (Table 1).A nearly normal frequency distribution of TN, PTN, SNS, SL, TGW, GL, and GW for the RILs was observed in each environment and the best linear unbiased estimate (BLUE) datasets with the absolute values of Skewness and Kurtosis coefficients approaching 0, except for TN and PTN obtained at 2020CP.(Table 1, Figure S1), suggesting the quantitative inheritance controlled by multiple loci.Awn lengths measured in the three positions on spikes showed a bimodal frequency distribution with the Kurtosis coefficients > 1, indicating the presence of major gene for these traits.
Correlation analysis demonstrated that the grain traits (TGW, GL, and GW) and the spike traits (SL and SNS) were positively correlated (r = 0.20-0.77,P < 0.01), but they were negatively correlated with TN and PTN (r = 0.14-0.55,P < 0.05) (Figure S2).TN and PTN were positively correlated with each other (r = 0.57, P < 0.01).Lengths of awn on the top, centre, and bottom of spikes were correlated (P < 0.01), and they were also correlated with GL, GW and SNS (P < 0.05).Except for TN, the other 7 traits were significantly correlated among different environments (P < 0.01) (Table S1).A significant interaction was observed between genotypes and environments for TN and PTN, while no such interaction was detected for the other traits (Table S2).

BSE-Seq analysis
The statistical parameters from the BSE-Seq analysis are summarized in Table S3.A total of 10,774 SNP variants were detected between the high-and low-TGW DNA pools (Bulk-HTGW and Bulk-LTGW).The most abundant enrichment of the TGW-associated SNPs and InDels was observed in the genomic intervals of 28.60-206.77Mb and 342.32-621.14Mb on chromosome 4B (8075) and 33.00-87.24Mb on chromosome 7 A (2699) of Chinese Spring (CS) reference genome sequence Ref-Seq v1.0 (Fig. 2c, Table S3).

BSR-Seq analysis of crowns and inflorescences
The statistical parameters of the RNA sequencing and comparisons between the RNA pools from contrasting tiller numbers (Bulk-HTN vs. Bulk-LTN) and between the RNA pools from contrasting spike lengths (Bulk-LS vs. Bulk-SS) are shown in Table S3.Raw reads of these RNA samples ranged from 135,474,852 to 156,857,352, and clean reads after trimming ranged from 129,072,674 to 149,787,196.The proportions of clean reads that were mapped to the CS reference genome RefSeq v1.0 were over 95%.The BSR-Seq analysis identified 1061 and 1508 SNP variants by comparing crown (Bulk-HTN vs. Bulk-LTN) and inflorescence (Bulk-LS vs. Bulk-SS) samples, respectively.Most SNP variants from tiller samples (Bulk-HTN vs. Bulk-LTN) were enriched on chromosome 4B (888), 6A (128), and 7A (45).SNPs from inflorescence samples (Bulk-LS vs. Bulk-SS) were mostly anchored on chromosomes 4B (1342), 4D (67), and 6A (99) (Fig. 2, Table S3).Most of the 464 common SNP variants detected in the tiller and inflorescence samples were located on chromosome 4B.

Construction of the genetic linkage map
The 16 K GBTS SNP array applied for the RIL population of Qingxinmai × 041133 generated 37,699 original SNPs.After removing those with the coverage depth < 5×, 14,868 SNPs were retained.Polymorphic SNPs between parents Qingxinmai and line 041133 were 4,939 in 2,430 loci.A total of 2,398 loci with clear positions on the reference genome were used to construct a genetic linkage map (3113.1 cM).Thirty linkage groups were established.Most chromosomes had single linkage groups, except for two for chromosomes 1D, 3D, 5D, 6B, and 7D and three for chromosomes 6A and 6D.The marker density of this  genetic linkage map was 3.27 cM per locus and 1.30 cM per marker.Subgenomes A, B, and D consisted of 344, 455, and 154 loci with the map distances of 1179.2, 1282.2, and 651.7 cM, respectively (Table S4).The genetic linkage maps constructed ranged from 50.9 cM (6D) to 197.3 cM (7A).Each chromosome contained 12 (4D) to 232 (3B) SNP markers.The average distance between adjacent SNPs and bin markers was in a range of 0.79 (3A) to 8.13 cM (3D) and 2.12 (2B) to 8.81 cM (3D).
The marker orders on most chromosomes were generally consistent with those in the CS reference genome sequence RefSeq v1.0 (Figure S3).The Chi-squared test revealed a genetic distortion in 377 polymorphic molecular markers (P < 0.05).Among them, 219 (58.09%) and 158 (41.91%) markers were biased towards line 041133 and Qingxinmai, respectively.Fifteen segregation distortion regions (SDR, ≥3SD loci) were detected in the RIL population.Eleven SDR originated from Qingxinmai and 4 from line 041133 (Table S5).

QTL mapping
Sixty QTL for the 8 traits investigated were detected on 18 chromosomes, except for 5D, 6D, and 7B.Nine QTL were detected in multiple environments and the BLUE data sets, explaining phenotypic variations ranging from 3.43 to 65.34%.Fifteen QTL were detected in one or two environments and the BLUE data sets, explaining phenotypic variations ranging from 1.53 to 15.27%.The resting 36 QTL were observed in single environments, explaining 3.15-19.41% of the phenotypic variations (Table 2, Figure S4-S9).
QAl.caas-4B for AL detected in 2021CP and the BLUE dataset was located in the same genetic interval as QSl.caas-4B/QSns.caas-4B.This QTL was contributed by 041133.But ALs on the top, central, and bottom of spikes appeared to be controlled by a major locus QAl.caas-5A.It was detected in all the three environments and the BLUE dataset, and explained the phenotypic variations of 59.96-65.34%(LOD = 56.12-71.09).The physical location of this locus was observed in a genomic interval of 688.17-697.64Mb (Table 2).The additive effect of QAl.caas-5A was provided by Qingxinmai.The SNP sequence of the closest molecular marker 5A_688174490 obtained by the 16 K GBTS SNP array was converted to a Kompetitive allele-specific PCR (KASP) marker KASP_ 5A_ 688174490 (Fig. 3, Table S6).This KASP marker proved to be tightly linked to QAl.caas-5A by genotyping the entire RIL population (Figure S10a).

Analysis of additive effects of the major QTL
Since the pleiotropic QTL for PTN, SL, SNS, TGW, GW, and GL were detected on chromosome 4B, we analyzed the additive effects of this locus on the corresponding traits using the BLUE datasets of the mapping population.Lines with the favorable alleles of QPtn/Sl/Sns/Tgw/ Gl/Gw.caas-4Bonly increased PTN, TGW, GL, and GW by 17.03%, 6.85%, 2.80%, and 4.23% over those without the alleles (Fig. 4).The addition of alleles at the minor loci QPtn.caas-2D,QTgw.caas-3D,QGw.caas-3D, and QGl.caas-3B further enhanced the performances of those traits by 20.79%, 11.26%, 4.45%, and 5.68%, respectively (Fig. 4).Lines with the favorable allele for SL at QSl.caas-4B only increased SL by 9.84% relative to lines without the favorable allele.The trait performances of SL and SNS appeared to be associated with the number of positive alleles.More favorable alleles increased the trait values by different magnitudes (Figure S11).

Analysis of the annotated genes in the target genomic interval of QTn/Ptn/Sl/Sns/Tgw/Gl/Gw.caas-4B
We analyzed the genes in the genomic region where the QTL on chromosome 4B resides using the in silico expression method and the RNA-Seq data generated from the BSR-Seq analysis with crowns and inflorescences.Twenty-two high confidential genes were annotated in the genomic interval of the pleiotropic QTL on chromosome 4B of the Chinese Spring reference genome RefSeq v1.0 (Table S7).The in silico expression of these annotated genes was analyzed in the Hexaploid Wheat Expression Database (IWGSC Annotation v1.1) assembled in the Triticeae Multi-Omics Center (http://202.194.139.32/).Five genes TraesC-S4B01G042300 (pleckstrin homology domain), TraesC-S4B01G042900 (ZnF), TraesCS4B01G043100 (Rht-B1b), TraesCS4B01G043400 (phytanoyl-CoA dioxygenase), and TraesCS4B01G044300 (microsomal glutathione S-transferase 3) were expressed in spikes and grains (Figure S12a).TraesCS4B01G042900, TraesCS4B01G043100, and TraesCS4B01G043300 were also differentially expressed between Qingxinmai and line 041133 in the BSR-Seq analysis with the crown and inflorescence RNA samples (Figure S12b).The expression of these genes in crowns and inflorescences of Qingxinmai and line 041133 were further determined by qPCR (Figure S13).Gene TraesC-S4B01G042900 was differentially expressed in crowns but not in inflorescences, TraesCS4B01G043100 (Rht-B1b) in both tissues, and TraesCS4B01G043300 in inflorescences but not in crowns.The expression of the three genes was higher in Qingxinmai than in line 041133, except TraesC-S4B01G043100 in crowns.

Discussion
We detected a pleiotropic locus on chromosome 4B using a RIL population derived from a wheat landrace Qingxinmai and a breeding line 041133.We further determined that the alleles of this locus from the two parents confer different traits.The allele from Qingxinmai was responsible for increasing TN and PTN.The allele from line 041133 increased spike traits (SL and SNS) and grain traits (TGW, GL, and GW), and even had a minor effect on increasing AL.The effects of this locus on the traits investigated were enhanced by several minor effective QTL, such as QPtn.caas-2D,QTgw.caas-3D,QGw.caas-3D, and QGl.caas-3B.
Many QTL for various wheat plant growth and yieldrelated traits have been characterized with different mapping populations and various types of molecular markers [7,32].Wheat chromosome 4B was associated with tiller number [22,33,34], spike length and spikelet number [35][36][37], and grain weight in separate studies [8,9,36].Yet some of those loci may not be localized in the same genomic regions of chromosome 4B.A QTL for tiller number was localized at 482.82 Mb [33].Four loci for PTN were located on the genomic regions of 75.74-640.97Mb [34,38].Liu et al. [38] and Deng et al. [39] identified a major QTL for PTN at 256.31 Mb.The allele of QTn/Ptn.caas-4B from Qingxinmai was anchored in the 28.95-32.17Mb genomic region, which appears to be different from the above loci for tiller number.But the genomic location of locus QTn/Ptn.caas-4Boverlaps with QPtn.sau-4B (28,941,377 and 32,167,076 bp) on chromosome 4B [22].QPtn.sau-4B was detected in Chuannong 16, a spring wheat cultivar developed in Sichuan province, which is unrelated to Qingxinmai.
Wheat spike traits are also associated with locus on chromosome 4BS.QSL.caas-4BS was mapped to a physical position of 25.80-46.60Mb in the Linmai 2 × Zhong 892 RIL population [36].The association of chromosome 4B and grain weight was reported in a RIL population of Doumai/Shi 4185 [36].In that study, one of the 11 QTL for grain weight QTkw.caas-4BS explained a high range of phenotypic variation (12.1-45.6%).That locus was located in a genomic region of 25.80-46.60Mb.A 483kb deletion in this region in Doumai contains genes ZnF, EamA, and Rht-B1 [8].In a most recent study, Song et al. [9] reported that wheat cultivar Heng 597 possessed a locus QTgw.cau-4B for grain weight.The deletion of approximately 500 kb fragment, also carrying these three genes, increased grain weight.The knockdown of Rht-B1b in Fielder increases plant height, spike length, and grain weight.But deletion of ZnF-B led to a slight reduction in grain size and plant height with no change in spike length compared to the wild-type Fielder.There is no large fragment deletion in the same genomic interval in Qingxinmai and line 041133 as in Doumai and Heng 597.Further study is needed to characterize the genes associated with the pleiotropic locus on chromosome 4B in the current study.
Segregation distortion of molecular markers is present in different genetic populations.Paillard et al. [45] detected 17% segregation distortion of RFLP and SSR markers in the RILs from cross between Arina and Forno wheats.We detected two large SDRs, one on chromosome 2B from Qingxinmai, and the other on chromosome 6B from line 041133.Two SDRs, SDR-4B.2 (26.9-30.8cM) and SDR-4B.3(30.8-34.4cM) from Qingxinmai, were associated with the pleiotropic locus QPtn.caas-4B for PTN.The SDRs may arise from chromosome recombination, gametophyte lethal genes, and segregation distortion factors [46,47].

Plant materials
Qingxinmai, a wheat landrace from Xinjiang, China, is characterized as long awn, slender spike, grain, and culm, and plenty of tillers (Fig. 1a).Line 041133 (pedigree: Jining 13/Tongmai 2) was developed in Qinghai province, with characteristics of tip-awns, thick spike, strong culm, fewer tillers, and larger grains (Fig. 1b).A RIL population consisting of 228 F 2:9 lines was developed by a single seed decent method from cross Qingxinmai × 041133 to be used as the mapping population.

Phenotype assessments
During the 2019-2020 and 2020-2021 wheat growing seasons, field plots were set for assessing traits of the mapping population and the parents in the experimental farms of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences in Beijing (2021BJ, 116.33°E, 39.96°N) and Changping, Beijing (2020CP, 2021CP, 116.26°E, 40.17°N),Zhaoxian, Hebei province (2020ZX, 114.78°E, 37.75°N), and Xinxiang, Henan province (2020XX, 113.98°E, 35.32°N), as well as a farm of Gansu Academy of Agricultural Sciences in Qingshui, Gansu province (2021QS, 105.80°E, 34.60°N).About 40 seeds of each line were planted in a one-row plot 2.0 m in length and a row spacing of 30 cm.A randomized complete block design with two replicates was used to arrange the RILs and the parents in each site.Field managements were performed according to the local practices for wheat production.Tiller traits, including TN at the tillering stage [Zadocks growth stage (GS) 31] [48] and PTN at the late milk stage (GS 77) were enumerated in 10 plants from each plot.At maturity, ten plants were randomly harvested from each plot to measure SL from the base of the rachis to the tip of terminal spikelet excluding awns and enumerate SNS.Length of awns at the top, central, and bottom of five spikes were measured.The phenotypic data of TGW, GL, and GW were measured by the Wanshen SC-G Automatic Seed Testing Analysis and Thousand Grain Weight Software (WSeen Inc., Hangzhou, China).

BSE-Seq analysis
Genomic DNA was isolated from grains with a cetyltrimethylammonium bromide method [49].Bulked DNA pools (Bulk-HTGW and Bulk-LTGW) were constructed by separately mixing equal amounts of DNA samples from 40 high-TGW (36.5-49.9g) and 40 low-TGW (20.4-29.0g) RILs.These DNA bulks, together with the parents, were subjected to exome capture sequencing on the WheatPanExomeV2 platform at Chengdu Teuni Technology (Chengdu, China).Uncaptured DNA fragments were removed, and the enriched exons were amplified by PCR.High-throughput DNA sequencing was performed on the Illumina platform (Illumina Inc., San Diego, CA, USA).SNPs obtained by mutation detection were filtered with the criteria of allele frequency < 0.3 or > 0.7 using the SNPindex algorithm to determine the genotype frequency of the extreme bulks [50].Significantly different SNP sites between the contrasting DNA bulks were statistically screened.Euclidean distance (ED) values of SNPs between the two DNA bulks were calculated using each allele depth with the quantile method.The ED values of SNP exceeding 99% was selected as the filtering threshold [51].

BSR-Seq analysis
Crown and inflorescence were sampled at GS 24 and GS 31 for RNA sequencing.Thirty phenotypically contrasting RILs each were chosen based on phenotypes of tiller and spike traits to construct high-and low-tiller number (Bulk-HTN and Bulk-LTN) and long-and short-spike (Bulk-LS and Bulk-SS) pools.A BSR-Seq analysis was performed following a previously described pipeline [52].In brief, RNA purified from the bulked samples with an Illumina TruSeq RNA sample preparation kit was sequenced on an Illumina Hiseq 4000 platform (Illumina Inc., San Diego, CA, USA).Adapter and low-quality sequences were truncated using Trimmomatic v0.36 [53].Highquality reads were aligned against the Chinese Spring (CS) reference genome sequence RefSeq v1.0 (http:// wheat-urgi.versailles.infa.fr)with the aid of STARv2.5.1b software [54].Single nucleotide polymorphism (SNP) variants [P < 1e-8 for the Fisher's Exact Test (FET) and the allele frequency difference (AFD) > 0.6] were identified from confident alignments using the "Haplotype Caller" module assembled in software GATK v3.6 [55].

QTL mapping
The RILs and their parents were genotyped with genomic DNA samples by the wheat 16 K GBTS SNP array (Mol-Breeding Biotechnology Co. Ltd., Shijiazhuang, China) (http://www.molbreeding.com).Raw reads generated were processed with fastp v0.20.0 [56] and then aligned to the CS reference genome RefSeq v1.0 with Burrows-Wheeler aligner [57].High-quality SNPs were obtained and filtered by GATK v3.5 [58], and SNP variants with the read depth < 5 were excluded from further analysis.SNP loci were classified as heterozygous genotypes when the SNP variation frequency ranged from 0.2 to 0.8, and the remaining genotypes were homozygous.Polymorphic SNPs between Qingxinmai and line 041133 were extracted for further analysis.A genetic linkage map was constructed for QTL calling using IciMapping 4.2 [59].Only one marker was selected as a delegate from each bin to construct the linkage map.QTL for the traits were detected by IciMapping 4.2 with the inclusive composite interval mapping (ICIM) method.A test of 1,000 permutations was used to identify the logarithm of odds (LOD) threshold (> 3.0) that corresponded to a genome-wide false discovery rate of 5% (P < 0.05).

KASP marker development and QTL validation
DNA sequences of selected SNPs were used to develop KASP markers with the help of the Triticeae Multi-omics Center (http://202.194.139.32).Primers were designed using PolyMarker (http://polymarker.tgac.ac.uk/).The probe sequences for the FAM and the HEX signals were separately added to the primers specific for the two parental genotypes.A thermal Cycler (C1000 Touch™, Bio-Rad, Foster City, CA, USA) was used to perform KASP assays.The reaction mixture (10 µl) was prepared by mixing 5 µl of 2× master mix (Wuhan Gentides Biotech Co., Ltd., Wuhan, China), 0.2 µl of primer mix, 3 µl of ddH 2 O, and 2 µl of DNA template (50-150 ng/µl).The thermal cycling profile included 94 °C for 15 min hotstart activation, a touchdown phase of 10 cycles (94 °C for 20 s, touchdown at 61 °C initially and then decreased by 0.6 °C per cycle for 60 s), and 26 cycles of regular PCR (94 °C for 20 s, 55 °C for 1 min).The following cycling and resting steps set at 94 °C for 20 s and 57 °C for 60 s (3-10 cycles per step) were performed if signals were poorly clustered.End-point fluorescence data were screened using the microplate reader FLUOstar Omega SNP (BMG Labtech, Durham, NC, USA) and analyzed by the Klustering Caller Software (http://www.lgcgroup.com/).The KASP markers polymorphic between the two parents were used to genotype the entire RIL population.

Statistical analysis
The BLUE value was calculated with the Aov [analysis of variance (ANOVA) of multi-environmental trials] function in the QTL IciMapping 4.2 [59] to be used for combined QTL detection, correlation, and normal distribution analyses.The H 2 for each trait were was also analyzed through QTL IciMapping 4.2.Phenotypic correlation was computed from the BLUE value of each line in SPSS v. 20.0 for Windows (IBM SPSS, Armonk, NY, USA).The Student's t-test was used to evaluate the significance of differences in SPSS at P < 0.05 or P < 0.01.

Expression of candidate genes in the target genomic region
Information on gene annotation in target genomic intervals was obtained with JBrowse in the Triticeae Multi-Omics Center (http://202.194.139.32).Gene codes annotated were used to predict the gene expression levels in the GeneExpression toolbar in the Triticeae Multi-Omics Center (http://202.194.139.32).Significantly differential expression was defined as an absolute log 2 value (fold change) > 1 at P < 0.01.
Total RNA was isolated from crowns (sampled at GS 31) and inflorescences (sampled at GS 41) of Qingxinmai and line 041133 by a FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China).The first-strand cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (https:// www.takarabiomed.com.cn/).Gene-specific quantitative real-time PCR (qPCR) primer pairs for candidate genes were designed according to the gene annotations from the CS reference genome RefSeq v1.0 [60].qPCR assays were performed on a BioRad CFX system with the Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China).Wheat Actin gene was amplified as the reference gene.Relative expression was determined using the 2 −ΔΔCT method [61].Three biological replicates were taken from crowns and inflorescences.Three replicates for each RNA sample were run as technical replicates.

AFD
The allele frequency difference AL Awn length Aov Analysis of variance (ANOVA) of multi-environmental trials B1 Tipped1 B2 Tipped2 BJ Beijing

Fig. 2
Fig. 2 Distribution of single nucleotide polymorphisms (SNPs) between the phenotypically contrasting bulks of the Qingxinmai × 041133 RIL population.(a) BSR-Seq analysis of the high-and low-tiller number RNA pools (Bulk-HTN and Bulk-LTN); (b) BSR-Seq analysis of the long-and short-spike RNA pools (Bulk-LS and Bulk-SS); and (c) exon trapping analysis of the high-and low-thousand-grain weight pools (Bulk-HTGW and Bulk-LTGW)

Fig. 3
Fig. 3 Genetic linkage map (a), QTL analysis method (b), and effects (c) of locus QAL.caas-5A on awn lengths.The BLUE values for awn lengths of the Qingxinmai × 041133 RILs were grouped based on the genotypes of the locus-specific KASP marker KASP_5A_ 688174490.**, P < 0.01

Fig. 4
Fig. 4 Additive effects of the QTL detected on productive tiller number (PTN) (a), thousand-grain weight (TGW) (b), grain width (GW) (c), and grain length (GL) (d) using the BLUE datasets of the Qingxinmai × 041133 RIL population.+ and −: presence and absence of the favorable alleles of the target QTL based on genotypes of the flanking markers of the corresponding QTL.**, P < 0.01.ns: no significant difference

Table 2 (
continued) environments, accounting for 3.15-19.41% of the phenotypic variations.Line 041133 contributed 16 positive alleles and Qingxinmai contributed the other 4 positive alleles (Table